Short Tandem Repeats (STRs) Detection for Fragile X Syndrome Diagnosis

Short Tandem Repeats (STRs) are short nucleotide sequences that are repeated many times throughout the genome. While most STRs are harmless and have no noticeable effect, some are located in functional regions of the genome. When these STRs have an unusual number of repeats, often too many, it can disrupt normal function and lead to genetic disorders such as Huntington disease, Myotonic Dystrophy Type 1, Frontotemporal dementia (FTD), Spinal Muscular Atrophy (SMA) and Fragile X Syndrome (FXS).



FXS and Fragile X associated disorders are characterized by a CGG trinucleotide repeat expansion in the 5’ UTR of the FMR1 (Familial Mental Retardation1) gene. This expansion can lead to a variety of consequences depending on the length of the CGG expansion. Repeat expansions in the FMR1 gene are associated with hypermethylation and inactivation of gene expression and subsequent loss of protein expression.



Individuals with fewer than 45 CGG repeats (normal) and between 45-54 CGG repeats (intermediate) in the FMR1 gene are typically unaffected by associated disorders. However, individuals with 55-199 repeats (premutation alleles) have a risk of passing on a full mutation to their offspring. This risk increases with the number of CGG repeats and the presence of AGG interruptions within the repeat sequence. Individuals with full mutation (~200 CGG repeats) typically have FXS. Fully expanded FMR1 alleles are often hypermethylated, silencing the gene and leading to the Fragile X phenotype. However, the severity of this phenotype can vary, influenced by the degree of methylation and mosaicism. Precisely determining repeat length and methylation status is crucial for accurate diagnosis and understanding the potential impact of FMR1-related disorders. Being an X-lined disorder, only the mother can pass on a full mutation to male offspring, while females can inherit full mutations from either parent.



Fluorescent Polymerase chain reaction (PCR) has several advantages in fragment analysis over southern blotting techniques. PCR is less laborious and has higher throughput. For larger repeat expansions, a Triplet-primed PCR is used that employs a primer binding to the repeat region and a limiting primer targeting the repeat itself. This generates multiple products, with the largest reflecting the expansion size. A third, abundant primer amplifies these products, and capillary electrophoresis is used for size analysis.



Over the years PCR technologies have emerged to navigate high GC rich regions and secondary structures, to make their way into diagnostics.





AmplideX® Fragile X Dx & Carrier Screen test from Asuragen, a bio-techne brand uses unique and proprietary Triplet Repeat Primed PCR (TP-PCR) to sensitively detect and identify the number of CGG repeats in the FMR1 gene using genomic DNA isolated from peripheral whole blood specimens. The test detects allele expansions including low abundance full mutation size mosaics with up to at least 1300 CGG repeats.



PCR free whole genome sequencing (WGS) emerged as an alternate diagnostic tool for identifying variants such as SNPs (single nucleotide polymorphisms), indels (insertions/deletions) and CNVs (copy number variations). But more complex structural variations such as STRs are more challenging. Current sequencing technologies struggle to accurately sequence long repeat tracts, such as the CGG repeats associated with several genetic disorders. Long-read sequencing can more accurately analyze regions of the genome with repetitive DNA sequences, which are often challenging to sequence with short-read technologies. Single Molecule, Real-Time (SMRT) sequencing by PacBio, offers a promising approach by leveraging long-read capabilities and single-molecule sequencing enabling the generation of long reads, often exceeding 20 kb, particularly useful for sequencing repeat-rich regions like the FMR1 gene.



Analyzing repeat expansion data is a complex task. Novel software algorithms offer accurate size determination across diverse technological platforms, empowering researchers to reliably diagnose complex genetic disorders.